Project Summary/Abstract Pancreatic adenocarcinoma (PDA) is the most lethal major cancer, with a 5-year survival rate below 10%. This is largely due to the lack of effective treatment options. The physiology and biochemical nature of pancreatic tumors is fundamental to this therapeutic resistance. PDA cells exist in a dense, fibrotic and thus, nutrient-depleted tumor microenvironment. Predictably, metabolism is reprogrammed in PDA cells to fuel their maintenance and growth. For example, our group recently described a non-canonical pathway utilized by PDA cells to mediate oxidative stress, thereby permitting their full growth potential. More recently, my preliminary results revealed that inhibition of malic enzyme 1 (ME1), the last enzyme in this metabolic pathway, can significantly blunt tumor growth in vitro and in vivo. There are three ME isoforms in mammals, ME1-3. I identified an inverse correlation between ME1 dependence and ME2 expression in PDA cells. In other words, ME1 dependent PDA cells have low/no ME2, and ME2 expressing PDA cells are resistant to ME1 inhibition. Intriguingly, nearly 50% of PDA express low/no ME2. ME2 is in close chromosomal proximity to SMAD4, a tumor suppressor lost in PDA. Based on their genomic location, co-loss of ME2 also frequently occurs. Therefore, the loss of ME2 provides a potential context for ME1-dependent synthetic lethality and a patient stratification method for ME1 inhibitors. There is therefore a fundamental need to understand the functional roles of MEs in order to exploit this unique therapeutic vulnerability in PDA. The working hypothesis of this project is that functional redundancy exists between cytosolic ME1 and mitochondrial ME2. When ME1 is impaired, ME2 provides metabolic/redox compensatory activity. Further, loss of ME2 expression provides a context for ME1-dependent synthetic lethality. This will be tested in two parts. (Aim 1) Mechanistically, the metabolic and subcellular functions of MEs in PDA growth will be defined. The metabolic roles of MEs will be examined by steady-state metabolomics, isotope tracing and flow cytometry following alteration of ME expression. The subcellular localization and potential physical interactions of MEs will also be investigated. (Aim 2) Functionally, the compensatory effect between ME1 and ME2 on PDA growth will be examined in vitro, ex vivo and in vivo using genetic knockdown/out and overexpression. These studies will contribute to our understanding of the mechanisms that regulate metabolism in PDA and the roles of MEs. Further they will pave the way for the development of ME1-targeted drugs for PDA, while also providing a strategy for patient stratification.